Cell-penetrating anti-DNA antibodies and uses thereof inhibit DNA repair

ABSTRACT

Antibodies that penetrate cell nuclei and inhibit DNA repair or interfere with DNA metabolism are provided for treatment of cancer (both directly and by sensitizing cancer cells to DNA-damaging treatments) or inhibiting or preventing viral infection, proliferation or metabolism. The method involves treating cells with a composition containing cell-penetrating anti-DNA antibodies or derivatives thereof, alone or in combination with treatment that induces DNA damage such as DNA-damaging chemotherapy or radiation. The impact of the cell-penetrating anti-DNA antibodies or derivatives thereof is potentiated in cancer cells that are deficient in DNA repair, and the cell-penetrating anti-DNA antibodies or derivatives thereof are synthetically lethal to cancer cells with DNA repair deficiencies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 application of the International ApplicationNo. PCT/US2012/031860, entitled “Cell-Penetrating Anti-DNA Antibodiesand Uses Thereof Inhibit DNA Repair” by James E. Hansen, Peter M.Glazer, Richard H. Weisbart, Robert N. Nishimura, and Grace Chan, filedon Apr. 2, 2012, which claims benefit of and priority to U.S.Provisional Application No. 61/470,918, filed Apr. 1, 2011, which ishereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under CA129186 awardedby the National Institutes of Health. The Government has certain rightsin the invention. This work was supported by the U.S. Department ofVeterans Affairs, and the Federal Government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention is generally related to the field of antibody therapy forthe treatment of cancer, and more particularly to the use ofcell-penetrating anti-DNA antibodies to inhibit DNA repair, sensitizecells to radiotherapy and DNA-damaging chemotherapy, and to selectivelykill cancer cells with pre-existing deficiencies in DNA repair.

BACKGROUND OF THE INVENTION

Most cancer therapies are severely limited by significant side effectsdue to non-specific tissue toxicity, and identification of novel agentsthat are selectively toxic to cancer cells or selectively sensitizetumors to treatment is a key goal in cancer research. A significantamount of work has focused on applying the specific binding activity ofmonoclonal antibodies to the development of tumor-specific therapies.Select antibodies such as trastuzumab (Herceptin®), rituximab(Rituxan®), and cetuximab (Erbitux®) have received approval for use inhuman cancer therapy, but all lack the ability to penetrate into cancercells and are therefore limited to attacking targets located on theexternal surface of tumor cells.

A significant number of tumor-specific targets are located inside cellsand nuclei, and numerous types of cancer are particularly vulnerable totreatments that inhibit DNA repair.

It is therefore an object of the invention to provide cell-penetratingantibodies, such as anti-DNA antibodies, that inhibit DNA repair.

It is a further object of the invention to provide compositions thatincrease the sensitivity of cancer cells to radiation therapy and/orchemotherapy.

It is a further object of the invention to provide cell-penetratingantibodies and derivatives thereof that are selectively toxic to canceror other undesirable cells with pre-existing deficiencies in DNA repair,typically associated with familial syndromes due to mutations in DNArepair genes but also occurring sporadically with silencing or amutation in DNA repair genes.

It is a further object of the invention to provide cell-penetratingantibodies and derivatives thereof that prevent or inhibit viralinfection, integration, and/or replication by perturbing host DNArepair.

SUMMARY OF THE INVENTION

Methods of using anti-DNA antibodies to penetrate cell nuclei andinhibit DNA repair have been developed. In preferred embodiments, thecell is a neoplasm, typically a cancer cell such as a carcinoma. Themethod of treating cancerous or certain infected cells involvescontacting the cells with the cell-penetrating anti-DNA antibodies,alone or in combination with other agents such as chemotherapeuticagents or radiation. In some embodiments, the method involves assaying asubject or tumor for one or more gene mutations or alterations in normalgene expression that impair DNA repair, then the anti-DNA antibodies areselected for use in treating the cells if the one or more mutations orpatterns of altered expression or function are identified.

Cells that have impaired DNA repair are particularly good targets forthis method. In preferred embodiments, the cells are defective in theexpression of or have a mutation in a gene involved in DNA repair, DNAdamage checkpoints, DNA synthesis, homologous recombination, ornon-homologous end-joining. Exemplary genes include XRCC1, ADPRT(PARP-1), ADPRTL2, (PARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2, FANCD2,PMS2, p53, p21, PTEN, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, MMS19,RAD51, RAD51b, RAD51C, RAD51D, DMC1, XRCCR, XRCC3, BRCA1, BRCA2, PALB2,RAD52, RAD54, RAD50, MRE11, NB51, WRN, BLM, KU70, KU80, ATM, ATR CHK1,CHK2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCC,FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, and RAD9. In someembodiments, the defective gene is a tumor suppressor gene. For example,the cells can have one or more mutations in BRCA1 or BRCA2.

Many cancer therapy procedures such as chemotherapy and radiotherapywork by overwhelming the capacity of the cell to repair DNA damage,resulting in cell death. In some embodiments, the cells are resistant toradiation therapy and/or chemotherapy. Methods are also provided forenhancing the efficacy of radiotherapy and/or chemotherapy in a subjectby administering to the subject a composition containingcell-penetrating anti-DNA antibodies. In some embodiments, theantibodies increase the cells' sensitivity to the radiation therapyand/or chemotherapy. The chemotherapeutics that can be enhanced by thismethod include those that damage DNA or inhibit DNA repair. The anti-DNAantibodies may be administered to the subject before, concurrently, orafter the administration of radiotherapy and/or chemotherapy. Inpreferred embodiments, the anti-DNA antibodies are administered to thesubject at least two days before or after radiotherapy and/orchemotherapy, more preferably at least one day before or afterradiotherapy and/or chemotherapy, and even more preferably concurrentlywith the radiotherapy and/or chemotherapy.

Cell-penetrating anti-DNA antibodies are disclosed that inhibit DNArepair. These antibodies are transported into the nucleus of the cellwithout the aid of a carrier or conjugate. The anti-DNA antibody can insome embodiments bind single stranded DNA (ssDNA), double-stranded DNA(dsDNA), or a combination thereof. Antibodies specific fordouble-stranded DNA (dsDNA) are present in 70% of patients with systemiclupus erythematosus (SLE), compared to 0.5% of people without SLE.Therefore, in some embodiments, the cell-penetrating anti-DNA antibodyis isolated or derived from a subject with SLE or an animal, such as amouse or rabbit, with a similar autoimmune condition, then humanized orexpressed recombinantly and administered as a dimer or single chainantibody.

Examples of useful cell-penetrating anti-DNA antibodies are themonoclonal anti-DNA antibody 3E10, or a variant or fragment thereof thatbinds the same epitope(s) as 3E10. In preferred embodiments, theanti-DNA antibody is a single chain variable fragment of an anti-DNAantibody, or conservative variant thereof. For example, the anti-DNAantibody can be a single chain variable fragment of 3E 10 (3E 10 scFv),or conservative variant thereof. The 3E10 scFv is preferably produced asa recombinant protein expressed from an expression vector in a mammaliancell, such yeast, e.g., Pichia pastoris.

In preferred embodiments, the antibodies have the same epitopespecificity as monoclonal antibody 3E10, produced by ATCC Accession No.PTA 2439 hybridoma. This can be achieved by producing a recombinantantibody that contains the paratope of monoclonal antibody 3E10.Alternatively, this can be achieved by creating a hybridoma fromlymphocytes isolated from a human subject or a mouse or otherexperimental animal with an autoimmune disease, such as SLE. Suitableantibodies include full-length antibodies, single chain antibodies, andantibody fragments. The antibody can also be a bispecific monoclonalantibody that specifically binds a second therapeutic target in thenucleus of the cell. For example, in some embodiments, the antibodyspecifically binds a protein in the nucleus of a cell such as a DNArepair protein, a DNA replication protein, a DNA damage responseprotein, a cell cycle regulatory protein, a DNA damage checkpointprotein, an apoptosis regulatory protein, or a stress response protein.Exemplary targets in these categories respectively include RAD52protein, ataxia telangiectasia mutated protein (ATM), CHK2 or CHK1proteins, BCL2 protein, heat shock protein 70 (HSP70), Myc protein, andRas protein.

In a preferred embodiment, the cell-penetrating anti-DNA antibodies areprovided in a unit dosage in an amount effective to inhibit DNA repairin a cancer, which may include a pharmaceutically acceptable excipientin the same vial or separately, in a kit, wherein the antibodies arepresent in an amount effective to inhibit DNA repair in a cancer cell.In preferred embodiments, the antibody is present in amount from about200 mg/m² to about 1000 mg/m², including about 200, 250, 300, 350, 400,450, 500, 600, 700, 800, 900, or 1000 mg/m². In some embodiments,pharmaceutical composition is in a unit dosage form for intravenousinjection. In some embodiments, the pharmaceutical composition is in aunit dosage form for intratumoral injection.

The pharmaceutical composition (e.g., dosage unit) can further contain,or be provided in a kit with, additional therapeutic agents. Forexample, the additional therapeutic agent can be an antineoplasticagent, a radiosensitizing agent, or a combination thereof. Preferably,the antineoplastic agent damages DNA or inhibits DNA repair. In someembodiments, the antineoplastic agent is cisplatin, cytoxan,doxorubicin, methotrexate, mitomycin e, nitrogen mustard, aribonucleotide reductase inhibitor (e.g., hydroxyurea), tirapazamine,temozolomide, or a topoisomerase inhibitor (e.g, camptothecin).

Non-limiting examples of radiosensitizers that can be present in thepharmaceutical composition include cisplatin, doxorubicin, gemcitabine,5-fluorouracil, PARP1 inhibitors, histone deacetylase inhibitors,proteasome inhibitors, epidermal growth factor receptor (EGFR)inhibitors, insulin-like growth factor-1 (IGF-1) receptor inhibitors,CHK1 inhibitors, mTOR inhibitors, kinase inhibitors, pentoxifylline, andvinorelbine.

The pharmaceutical composition (e.g., dosage unit) can further containone or more therapeutic monoclonal antibodies for treating cancer. Inpreferred embodiments, the therapeutic monoclonal antibody isbevacizumab, cetuximab, rituximab, trastuzumab, or a combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of the 3E10 single chain antibody variablefragment (3E10 scFv) composed of the variable regions of the 3E10 lightand heavy chains joined by a linking domain (LD). Myc and His6 tags wereadded to allow for detection and purification. 3E10 scFv penetrates intothe nuclei of cancer cells (demonstrated with Skov-3 ovarian cancercells treated with 5 μM 3E10 scFv for 30 minutes, and then fixed andstained with an anti-Myc antibody). FIG. 1B is a bar graph showingclonogenic survival (surviving fraction relative to control) of U251human glioma cells irradiated at 0 Gy (bars 1-2) or 4 Gy (bars 3-4) inthe presence of control buffer (bars 1 and 3) or 10 μM 3E10 Fv (bars 2and 4). Error bars represent standard error for replicate experiments.FIGS. 1C-1D are graphs showing cell death (%, measured by propidiumiodide fluorescence) of U87 human glioma cells as a function ofdoxorubicin (Dox) (0-250 nM) (FIG. 1E) or paclitaxel (0-2.5 nM) (FIG.1F) in the presence (dashed line) or absence (solid line) of 10 μM 3E10scFv.

FIG. 2A demonstrates the different conformations of DNA substrates usedin 3E10-DNA binding experiments. FIG. 2B is a graph showing fraction (%)of radiolabeled oligonucleotides with (solid line) or without (dashedline) free single-strand tail bound by 3E10 (determined by gel mobilityshift analyses) after incubation with increasing concentrations of 3E10(0-1 μM). These binding curves yield K_(d)s of 0.2 μM and 0.4 μM for3E10 binding to substrates with and without a free single-strand end,respectively. FIGS. 2C-2H present individual 3E10-DNA binding curves foreach DNA conformation. FIG. 2I is a bar graph showing single-strandbreak/base excision repair (BER) (% n+1 products represent theproportion of incompletely repaired intermediates in which a nucleotidehas been added to a gapped duplex molecule but the remaining singlestrand break has not been repaired to yield a full length product) insynthetic radiolabeled duplex DNA substrates incubated with requisiterepair enzymes in the presence of control buffer (open bars) or 20 μME10 (solid bars). The repair reaction was stopped at the indicated timepoints, and the n, n+1, and duplex reaction products were quantified bygel electrophoresis and autoradiography.

FIG. 3A is a schematic for an in vitro DNA strand exchange assay. FIGS.3B and 3C are bar graphs showing the impact of increasing dose of 3E10(0-35 μM) on RAD51-mediated strand exchange using wild-type hRAD51protein (FIG. 3B) or variant, hRAD51K133R (FIG. 3C). ThehRAD51K133R-variant is even more active for strand exchange than thewild-type protein because it does not hydrolyze ATP. 3E10 inhibitsstrand exchange by both wild-type RAD51 and the hRAD51K133R variant.Immunofluorescence images demonstrate DNA double strand breaks (γH2AXfoci) per U251 glioma cell 24 hours after irradiation with 2 Gy in thepresence of control buffer or 10 μM E10 scFv. FIG. 3D is a bar graphshowing the average number of DNA double strand breaks (γH2AX foci) perU251 glioma cell 24 hours after irradiation with 2 Gy in the presence ofcontrol buffer (open bar) or 10 μM 3E10 scFv (solid bar). Error barsrepresent standard errors.

FIGS. 4A-4B are bar graphs showing clonogenic survival (survivingfraction relative to control) of BRCA2-proficient (FIG. 4A) orBRCA2-deficient (FIG. 4B) human ovarian cancer cells treated withcontrol buffer (open bars) or 10 μM 3E10 scFv (solid bars) by colonyformation measured 1-2 weeks following treatment. FIG. 4C is a graphshowing % cell death of BRCA2-deficient (PEO1) and BRCA2-proficient(PEO4) human ovarian cancer cells treated with 3E10 scFv (0-2 μM). Theimpact of 3E10 scFv on cell survival was evaluated three days aftertreatment by CellTiterGlo® luminescence, which reports ATP levels as ameasure of metabolically active cells. Error bars represent standarderror of the mean of six measurements. FIG. 4D is a graph showing theclonogenic survival (surviving fraction relative to control measured bycolony formation) of BRCA2-deficient CAPAN1/neo cells (human pancreaticcancer cells) treated with control buffer or 3E10 scFv. FIG. 4E is agraph showing cell death (%) of BRCA2-deficient (dashed line) orBRCA2-proficient (solid line) human ovarian cancer cells treated with 0μM, 2.5 μM, 5 μM, or 10 μM of the full 3E10 antibody. FIGS. 4F and 4Gare bar graphs showing cell death (%) of BRCA2-deficient (FIG. 4G) orBRCA2-proficient (FIG. 4F) human ovarian cancer cells treated withcontrol buffer (bar 1), 10 μM 3E10 (bar 2), 3 nM doxorubicin (Dox) (bar3), or 10 μM 3E10+3 nM doxorubicin (bar 4).

FIG. 5 is a bar graph showing tumor volume (% increase) of U87 gliomatumors generated in SCID mice by subcutaneous injection treated byintraperitoneal injection of control buffer (bar 1), 3E10 antibody alone(0.8 mg in 0.5 mL PBS, 10 μM) (bar 2), doxorubicin (Dox) alone (80μg/kg) (bar 3), or both 3E10 and doxorubicin (bar 4). Each treatmentgroup was composed of 4 mice. The impact of treatment was evaluated bymeasuring tumor growth three days after injection.

FIG. 6A is a graph showing tumor volume (mm³) as a function of time(days) of human glioma xenograft mice treated with intraperitonealinjection of control PBS buffer (solid diamonds and triangles) or 3E10(1 mg in PBS) (open diamonds and triangles) twenty-six days after U87cell implantation (tumors had grown to a mean size of approximately 100mm³), again 24 hours later, and then irradiated with 0 Gy (diamonds) or8 Gy (triangles) 2 hours after the second injection.

FIG. 6B presents Kaplan-Meier plots of progression-free survival in eachgroup. Progression-free survival is defined as survival with tumor nothaving increased in size by threefold or greater relative to baselinesize. Baseline size is defined as tumor size one day prior to antibodytreatment, which is represented as day 25 in FIG. 6A and day 0 in FIG.6B. Tumor tripling time (time required for tumors to increase in volumethreefold over baseline) was 9.5±0.5 days in tumors treated with 8 Gy ascompared to 13.7±1.8 days in tumors treated with 8 Gy+3E10 (p=0.04).3E10 alone, however, had no impact on U87 tumors relative to controlbuffer alone, with tumor tripling time of control tumors 6.8±0.7 daysversus 6.5±0.3 days in tumors treated with 3E10 alone (p=0.67).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “antibody” refers to natural or synthetic antibodies thatselectively bind a target antigen. The term includes polyclonal andmonoclonal antibodies. In addition to intact immunoglobulin molecules,also included in the term “antibodies” are fragments or polymers ofthose immunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules that selectively bind the target antigen.

The term “cell-penetrating anti-DNA antibody” refers to an antibody thatis transported into the nucleus of living mammalian cells andspecifically binds DNA (e.g., single-stranded and/or double-strandedDNA). In preferred embodiments, the antibody is transported into thenucleus of the cells without the aid of a carrier or conjugate. In otherembodiments, the antibody is conjugated to a cell-penetrating moiety,such as a cell penetrating peptide.

The term “specifically binds” refers to the binding of an antibody toits cognate antigen (for example DNA) while not significantly binding toother antigens. Preferably, an antibody “specifically binds” to anantigen with an affinity constant (Ka) greater than about 10⁵ mol⁻¹(e.g., 10⁶ mol⁻¹, 10⁷ mol⁻¹, 10⁸ mol⁻¹, 10⁹ mol⁻¹, 10¹¹ mol⁻¹, and 10¹²mol⁻¹ or more) with that second molecule.

The term “monoclonal antibody” or “MAb” refers to an antibody obtainedfrom a substantially homogeneous population of antibodies, i.e., theindividual antibodies within the population are identical except forpossible naturally occurring mutations that may be present in a smallsubset of the antibody molecules.

The term “DNA repair” refers to a collection of processes by which acell identifies and corrects damage to DNA molecules. Single-stranddefects are repaired by base excision repair (BER), nucleotide excisionrepair (MER), or mismatch repair (MMR). Double-strand breaks arerepaired by non-homologous end joining (NHEJ), microhomology-mediatedend joining (MMEJ), or homologous recombination. After DNA damage, cellcycle checkpoints are activated, which pause the cell cycle to give thecell time to repair the damage before continuing to divide. Checkpointmediator proteins include BRCA1, MDC1, 53BP1, p53, ATM, ATR, CHK1, CHK2,and p21.

The term “impaired DNA repair” refers to a state in which a mutated cellor a cell with altered gene expression is incapable of DNA repair or hasreduced activity of one or more DNA repair pathways or takes longer torepair damage to its DNA as compared to a wild type cell.

The term “chemosensitivity” refers to the relative susceptibility ofcancer cells to the effects of anticancer drugs. The more chemosensitivea cancer cell is, the less anticancer drug is required to kill thatcell.

The term “radiosensitivity” refers to the relative susceptibility ofcells to the harmful effect of ionizing radiation. The moreradiosensitive a cell is, the less radiation that is required to killthat cell. In general, it has been found that cell radiosensitivity isdirectly proportional to the rate of cell division and inverselyproportional to the cell's capacity for DNA repair.

The term “radioresistant” refers to a cell that does not die whenexposed to clinically suitable dosages of radiation.

The term “neoplastic cell” refers to a cell undergoing abnormal cellproliferation (“neoplasia”). The growth of neoplastic cells exceeds andis not coordinated with that of the normal tissues around it. The growthtypically persists in the same excessive manner even after cessation ofthe stimuli, and typically causes formation of a tumor.

The term “tumor” or “neoplasm” refers to an abnormal mass of tissuecontaining neoplastic cells. Neoplasms and tumors may be benign,premalignant, or malignant.

The term “cancer” or “malignant neoplasm” refers to a cell that displaysuncontrolled growth, invasion upon adjacent tissues, and oftenmetastasis to other locations of the body.

The term “antineoplastic” refers to a composition, such as a drug orbiologic, that can inhibit or prevent cancer growth, invasion, and/ormetastasis.

The term “anti-cancer moiety” refers to any agent, such as a peptide,protein, nucleic acid, or small molecule, which can be combined with thedisclosed anti-DNA antibodies to enhance the anti-cancer properties ofthe antibodies. The term includes antineoplastic drugs, antibodies thatbind and inhibit other therapeutic targets in cancer cells, andsubstances having an affinity for cancer cells for directed targeting ofcancer cells.

The term “virally transformed cell” refers to a cell that has beeninfected with a virus or that has incorporated viral DNA or RNA into itsgenome. The virus can be an acutely-transforming or slowly-transformingoncogenic virus. In acutely transforming viruses, the viral particlescarry a gene that encodes for an overactive oncogene calledviral-oncogene (v-one), and the infected cell is transformed as soon asv-one is expressed. In contrast, in slowly-transforming viruses, thevirus genome is inserted near a proto-oncogene in the host genome.Exemplary oncoviruses include Human papillomaviruses (HPV), Hepatitis B(HBV), Hepatitis C(HCV), Human T-lymphotropic virus (HTLV), Kaposi'ssarcoma-associated herpesvirus (HHV-8), Merkel cell polyomavirus,Epstein-Barr virus (EBV), Human immunodeficiency virus (HIV), and Humancytomegalovirus (CMV).

A virally infected cell refers to a cell that has been exposed to orinfected with a virus or carries viral genetic material, either RNA orDNA. The virus can be an oncogenic virus or a lytic virus or a latentvirus and can cause cancer, immunodeficiency, hepatitis, encephalitis,pneumonitis, respiratory illness, or other disease condition. it haspreviously been shown that retorviruses, specifically HIV, rely in partupon the base excision repair (BER) pathway for integration into hostDNA. The ability of 3E10 to inhibit DNA repair provides a mechanismwhereby 3E10 and other anti-DNA antibodies can ameliorate virally causeddiseases, in particular, by interfering with DNA repair and thereby byblocking the DNA or RNA metabolism that is part of virus life cycles aswell as part of viral infection of a cell. The examples demonstrate that3E10 inhibits the BER pathway, supporting efficacy in suppressinginfectivity of such retroviruses.

The term “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient.

The term “therapeutically effective” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination. Atherapeutically effective amount of a composition for treating cancer ispreferably an amount sufficient to cause tumor regression or tosensitize a tumor to radiation or chemotherapy.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “inhibit” means to decrease an activity, response, condition,disease, or other biological parameter. This can include, but is notlimited to, the complete ablation of the activity, response, condition,or disease. This may also include, for example, a 10% reduction in theactivity, response, condition, or disease as compared to the native orcontrol level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70,80, 90, 100%, or any amount of reduction in between as compared tonative or control levels.

A “fusion protein” refers to a polypeptide formed by the joining of twoor more polypeptides through a peptide bond formed between the aminoterminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein can be formed by the chemical couplingof the constituent polypeptides or it can be expressed as a singlepolypeptide from a nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone. Fusion proteins can beprepared using conventional techniques in molecular biology to join thetwo genes in frame into a single nucleic acid sequence, and thenexpressing the nucleic acid in an appropriate host cell under conditionsin which the fusion protein is produced.

II. Compositions

A. Anti-DNA Antibodies

Cell penetrating anti-DNA antibodies are disclosed for use in enhancingsensitivity of targeted cells to chemotherapy and/or radiation.Autoantibodies to single or double stranded deoxyribonucleic acid (DNA)are frequently identified in the serum of patients with systemic lupuserythematosus (SLE) and are often implicated in disease pathogenesis.Therefore, in some embodiments, anti-DNA antibodies can be derived orisolated from patients with SLE. In preferred embodiments, the anti-DNAantibodies are monoclonal antibodies, or fragments or variants thereof.The presence of circulating autoantibodies reactive against DNA(anti-DNA antibodies) is a hallmark laboratory finding in patients withsystemic lupus erythematosus (SLE). Although the precise role ofanti-DNA antibodies in SLE is unclear, it has been suggested that theantibodies play an active role in SLE pathophysiology. In the early1990s a murine lupus anti-DNA antibody, 3E10, was tested in experimentalvaccine therapy for SLE. These efforts were aimed at developinganti-idiotype antibodies that would specifically bind anti-DNA antibodyin SLE patients. However, 3E10 was serendipitously found to penetrateinto living cells and nuclei without causing any observed cytotoxicity(Weisbart R H, et al. J Immunol. 1990 144(7): 2653-2658; Zack D J, etal. J Immunol. 1996 157(5): 2082-2088). Studies on 3E10 in SLE vaccinetherapy were then supplanted by efforts focused on development of 3E10as a molecular delivery vehicle for transport of therapeutic moleculesinto cells and nuclei. Other antibodies were also reported to penetratecells.

The 3E10 antibody and its single chain variable fragment (3E10 scFv)penetrate into cells and nuclei and have proven capable of transportingtherapeutic protein cargoes attached to the antibody either throughchemical conjugation or recombinant fusion. Protein cargoes delivered tocells by 3E10 or 3E10 scFv include catalase, p53, and Hsp70 (Weisbart RH, et al. J Immunol. 2000 164: 6020-6026; Hansen J E, et al. Cancer Res.2007 Feb. 15; 67(4): 1769-74; Hansen J E, et al. Brain Res. 2006 May 9;1088(1): 187-96). 3E10 scFv effectively mediated delivery of Hsp70 toneurons in vivo and this resulted in decreased cerebral infarct volumesand improved neurologic function in a rat stroke model (Zhan X, et al.Stroke. 2010 41(3): 538-43).

It has now been discovered that 3E10 and 3E10 scFv, without beingconjugated to any therapeutic protein, enhance cancer cellradiosensitivity and chemosensitivity and that this effect ispotentiated in cells deficient in DNA repair. Moreover, 3E10 and 3E10scFv are selectively lethal to cancer cells deficient in DNA repair evenin the absence of radiation or chemotherapy. The Food and DrugAdministration (FDA) has established a pathway for the development ofmonoclonal antibodies into human therapies, and 3E10 has already beenapproved by the FDA for use in a Phase I human clinical trial designedto test the efficacy of 3E10 in experimental vaccine therapy for SLE(Spertini F, et al. J Rheumatol. 1999 26(12): 2602-8). Therefore, thesetypes of antibodies can be used for targeted therapy for cancersdeficient in DNA repair as well as in new targeted therapies for cancerto potentiate other cancer treatment modalities in cancers proficient inDNA repair as well as in cancers deficient in DNA repair.

Other cell-penetrating antibodies are known, both naturally occurring inSLE patients, and obtained by screening of antibody libraries orderivatization of known cell penetrating antibodies. In someembodiments, anti-DNA antibodies are conjugated to a cell-penetratingmoiety, such as a cell penetrating peptide, to facilitate entry into thecell and transport to the nucleus. Examples of cell-penetrating peptidesinclude, but are not limited to, Polyarginine (e.g., R₉), Antennapediasequences, TAT, HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II,Transportan, MAP (model amphipathic peptide), K-FGF, Ku70, Prion, pVEC,Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol,and BGTC (Bis-Guanidinium-Tren-Cholesterol). In other embodiments, theantibody is modified using TransMabs™ technology (InNexus Biotech.,Inc., Vancouver, BC).

In preferred embodiments, the anti-DNA antibody is transported into thenucleus of the cells without the aid of a carrier or conjugate. Forexample, the monoclonal antibody 3E10 and active fragments thereof thatare transported in vivo to the nucleus of mammalian cells withoutcytotoxic effect are disclosed in U.S. Pat. Nos. 4,812,397 and 7,189,396to Richard Weisbart, describing 3E10 antibodies and methods of producingand modifying 3E10 antibodies. Briefly, the antibodies may be preparedby fusing spleen cells from a host having elevated serum levels ofanti-DNA antibodies (e.g., MRL/1pr mice) with myeloma cells inaccordance with known techniques or by transforming the spleen cellswith an appropriate transforming vector to immortalize the cells. Thecells may be cultured in a selective medium and screened to selectantibodies that bind DNA.

Antibodies that can be used include whole immunoglobulin (i.e., anintact antibody) of any class, fragments thereof, and synthetic proteinscontaining at least the antigen binding variable domain of an antibody.The variable domains differ in sequence among antibodies and are used inthe binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a beta-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the beta-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies. Therefore, theantibodies contain at least the components of the CDRs necessary topenetrate cells, maintain DNA binding and/or interfere with DNA repair.The variable region of 3E10 contains all three properties.

Also disclosed are fragments of antibodies which have bioactivity. Thefragments, whether attached to other sequences or not, includeinsertions, deletions, substitutions, or other selected modifications ofparticular regions or specific amino acids residues, provided theactivity of the fragment is not significantly altered or impairedcompared to the non-modified antibody or antibody fragment.

Techniques can also be adapted for the production of single-chainantibodies specific to an antigenic protein. Methods for the productionof single-chain antibodies are well known to those of skill in the art.A single chain antibody can be created by fusing together the variabledomains of the heavy and light chains using a short peptide linker,thereby reconstituting an antigen binding site on a single molecule.Single-chain antibody variable fragments (scFvs) in which the C-terminusof one variable domain is tethered to the N-terminus of the othervariable domain via a 15 to 25 amino acid peptide or linker have beendeveloped without significantly disrupting antigen binding orspecificity of the binding. The linker is chosen to permit the heavychain and light chain to bind together in their proper conformationalorientation.

The anti-DNA antibodies can be modified to improve their therapeuticpotential. For example, in some embodiments, the cell-penetratinganti-DNA antibody is conjugated to another antibody specific for asecond therapeutic target in the nucleus of the cancer cell. Forexample, the cell-penetrating anti-DNA antibody can be a fusion proteincontaining 3E10 scFv and a single chain variable fragment of amonoclonal antibody that specifically binds the second therapeutictarget. In other embodiments, the cell-penetrating anti-DNA antibody isa bispecific antibody having a first heavy chain and a first light chainfrom 3E10 and a second heavy chain and a second light chain from amonoclonal antibody that specifically binds the second therapeutictarget.

Divalent single-chain variable fragments (di-scFvs) can be engineered bylinking two scFvs. This can be done by producing a single peptide chainwith two VH and two VL regions, yielding tandem scFvs. ScFvs can also bedesigned with linker peptides that are too short for the two variableregions to fold together (about five amino acids), forcing scFvs todimerize. This type is known as diabodies. Diabodies have been shown tohave dissociation constants up to 40-fold lower than correspondingscFvs, meaning that they have a much higher affinity to their target.Still shorter linkers (one or two amino acids) lead to the formation oftrimers (triabodies or tribodies). Tetrabodies have also been produced.They exhibit an even higher affinity to their targets than diabodies.

The therapeutic function of the antibody can be enhanced by coupling theantibody or a fragment thereof with a therapeutic agent. Such couplingof the antibody or fragment with the therapeutic agent can be achievedby making an immunoconjugate or by making a fusion protein, or bylinking the antibody or fragment to a nucleic acid such as an siRNA orto a small molecule, comprising the antibody or antibody fragment andthe therapeutic agent.

A recombinant fusion protein is a protein created through geneticengineering of a fusion gene. This typically involves removing the stopcodon from a cDNA sequence coding for the first protein, then appendingthe cDNA sequence of the second protein in frame through ligation oroverlap extension PCR. The DNA sequence will then be expressed by a cellas a single protein. The protein can be engineered to include the fullsequence of both original proteins, or only a portion of either. If thetwo entities are proteins, often linker (or “spacer”) peptides are alsoadded which make it more likely that the proteins fold independently andbehave as expected.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Antibodyhumanization techniques generally involve the use of recombinant DNAtechnology to manipulate the DNA sequence encoding one or morepolypeptide chains of an antibody molecule.

In some embodiments, the cell-penetrating antibody is modified to alterits half-life. In some embodiments, it is desirable to increase thehalf-life of the antibody so that it is present in the circulation or atthe site of treatment for longer periods of time. For example, where theanti-DNA antibodies are being used alone to treat cancer, e.g., cancercells having impaired DNA repair, it may be desirable to maintain titersof the antibody in the circulation or in the location to be treated forextended periods of time. In other embodiments, the half-life of theanti-DNA antibody is decreased to reduce potential side effects. Forexample, where the antibody is being used in conjunction withradiotherapy or chemotherapy, the antibody is preferably present in thecirculation at high doses during the treatment with radiation orantineoplastic drug but is otherwise quickly removed from thecirculation. Antibody fragments, such as 3E 10 scFv, are expected tohave a shorter half-life than full size antibodies. Other methods ofaltering half-life are known and can be used in the described methods.For example, antibodies can be engineered with Fc variants that extendhalf-life, e.g., using Xtend™ antibody half-life prolongation technology(Xencor, Monrovia, Calif.).

B. Cancers and Virally Transformed Cells

The antibodies can be used to treat cells undergoing unregulated growth,invasion, or metastasis. Cancer cells that have impaired DNA repair areparticularly good targets for cell-penetrating anti-DNA antibodies. Insome embodiments, the cell-penetrating anti-DNA antibodies are lethal tocells with impaired DNA repair. In preferred embodiments, the cells aredefective in the expression of a gene involved in DNA repair, DNAsynthesis, or homologous recombination. Exemplary genes include XRCC1,ADPRT (PARP-1), ADPRTL2, (PARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2,FANCD2, PMS2, p53, p21, PTEN, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF,MMS19, RAD51, RAD51B, RAD51C, RAD51D, DMC1, XRCCR, XRCC3, BRCA1, BRCA2,PALB2, RAD52, RAD54, RAD50, MRE11, NB51, WRN, BLM, KU70, KU80, ATM, ATRCHK1, CHK2, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG,FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, RAD1, and RAD9. In someembodiments, the defective gene is a tumor suppressor gene. In preferredembodiments, the cells have one or more mutations in BRCA1 or BRCA2.Gene mutations, such as BRCA1 and BRCA2 mutations, can be identifiedusing standard PCR, hybridization, or sequencing techniques.

Therefore, in some embodiments, the anti-DNA antibodies can be used totreat cancers arising from DNA repair deficient familial syndromes, suchas breast, ovarian, and pancreatic cancers. In these embodiments, theanti-DNA antibodies can be effective without radiotherapy orchemotherapy. For example, the anti-DNA antibodies can be used to treatcancers that are linked to mutations in BRCA1, BRCA2, PALB2, OR RAD51B,RAD51C, RAD51D or related genes. The anti-DNA antibodies can also beused to treat colon cancers, endometrial tumors, or brain tumors linkedto mutations in genes associated with DNA mismatch repair, such as MSH2,MLH1, PMS2, and related genes. The anti-DNA antibodies can also be usedto treat cancers with silenced DNA repair genes, such as BRCA1, MLH1, ORRAD51B, RAD51C, OR RAD51D. In these preferred embodiments, the abilityof the anti-DNA antibodies to inhibit DNA repair combined with theinherent repair deficiencies of these cancers can be sufficient toinduce cell death.

Therefore, in some embodiments, the anti-DNA antibodies can be used totreat virally transformed cells, such as cells infected with anoncovirus. Viral transformation can impose phenotypic changes on cell,such as high saturation density, anchorage-independent growth, loss ofcontact inhibition, loss of orientated growth, immortalization, anddisruption of the cell's cytoskeleton. The persistence of at least partof the viral genome within the cell is required for cell transformation.This may be accompanied by the continual expression from a number ofviral genes, such as oncogenes. These genes may interfere with a cell'ssignaling pathway causing the observed phenotypic changes of the cell.In some cases, the viral genome is inserted near a proto-oncogene in thehost genome. The end result is a transformed cell showing increased celldivision, which is favorable to the virus. In some embodiments, viraltransformation, viral infection, and/or metabolism is dependent upon DNArepair mechanisms. In these embodiments, inhibition of DNA repair usingthe disclosed anti-DNA antibodies also inhibits viral transformation,viral infection and/or metabolism in the cell.

In some embodiments, viral transformation, viral infection, and/ormetabolism is dependent upon metabolism of the virally encoded RNA orDNA as a part of the virus life cycle, producing intermediates subjectto binding and/or inhibition by 3E10 or other anti-DNA antibodies. Inthese embodiments, treatment with the disclosed anti-DNA antibodies alsoinhibits viral transformation, viral infection and/or metabolism in thecell.

Lentiviruses (such as HIV) have been previously found to be dependent onhost BER activity for infection and integration (Yoder et al., PLoS One,2011 Mar. 6(3) e17862). In addition, the ataxia-telangiectesia-mutated(ATM) DNA-damage response appears to be critical to HIV replication (Lauet al., Nat Cell Biol, 2005 7(5): 493-500). In some embodiments,retroviral (including lentiviruses, HIV) infection and integration isdependent on host DNA repair mechanisms. In these embodiments treatmentwith the disclosed anti-DNA antibodies also suppresses viralinfection/integration and suppresses re-infection in the viral lifecycle.

In some embodiments, lentiviral (HIV) replication is dependent on DNArepair. In these embodiments treatment with the disclosed anti-DNAantibodies also suppresses viral replication and suppresses re-infectionin the viral life cycle. Therefore, anti-DNA antibodies can be used totreat cells infected with a virus, such as an oncovirus. In someembodiments, antibodies inhibit viral transformation, replication,metabolism, or a combination thereof. Exemplary oncoviruses that can beaffected by anti-DNA antibodies include Human papillomaviruses (HPV),Hepatitis B (HBV), Hepatitis C (HCV), Human T-lymphotropic virus (HTLV),Kaposi's sarcoma-associated herpesvirus (HHV-8), Merkel cellpolyomavirus, Epstein-Barr virus (EBV), Human immunodeficiency virus(HIV), and Human cytomegalovirus (CMV). Anti-DNA antibodies may also beused to treat a latent virus. In some embodiments, the failure ofinfected cells to mount a DNA damage response to viruses, such as HSV-1,contribute to the establishment of latency. These virally infected cellstherefore have impaired DNA repair and are susceptible to treatment withanti-DNA antibodies. Exemplary latent viruses include CMV, EBV, Herpessimplex virus (type 1 and 2), and Varicella zoster virus.

Anti-DNA antibodies may also be used to treat active viral infectionsdue to viruses that give rise to cancer, immunodeficiency, hepatitis,encephalitis, pneumonitis, respiratory illness, or other diseasecondition, by virtue of the antibodies' ability to bind to DNA and tointerfere with DNA repair or RNA metabolisms that is part of the viruslife cycle.

Representative viruses whose life cycle or symptoms of the resultinginfection, that may be affected by administration of the antibodiesinclude Human papillomaviruses (HPV), Hepatitis B (HBV), HepatitisC(HCV), Human T-lymphotropic virus (HTLV), Kaposi's sarcoma-associatedherpesvirus (HHV-8), Merkel cell polyomavirus, Epstein-Barr virus (EBV),Human immunodeficiency virus (HIV), and Human cytomegalovirus (CMV).

Additional viruses that may be affected by administration of theantibodies include parvovirus, poxvirus, herpes virus, and other DNAviruses:

Virion Nucleic Examples (common naked/ Capsid acid Virus Family names)enveloped Symmetry type Group 1. Adenoviridae Adenovirus, NakedIcosahedral ds I Infectious canine hepatitis virus 2. PapillomaviridaePapillomavirus Naked Icosahedral ds I circular 3. ParvoviridaeParvovirus B19, Naked Icosahedral ss II Canine parvovirus 4.Herpesviridae Herpes simplex Enveloped Icosahedral ds I virus,varicella-zoster virus, cytomegalovirus, Epstein-Barr virus 5.Poxviridae Smallpox virus, cow Complex Complex ds I pox virus, sheep poxcoats virus, orf virus, monkey pox virus, vaccinia virus 6.Hepadnaviridae Hepatitis B virus Enveloped Icosahedral circular, VIIpartially ds 7. Polyomaviridae Polyoma virus; JC Naked Icosahedral ds Ivirus (progressive circular multifocal leukoencephalopathy) 8.Anelloviridae Torque teno virus

RNA viruses that may be affected by administration of the antibodiesinclude:

Nucleic Examples (common Capsid Capsid acid Virus Family names)naked/enveloped Symmetry type Group 1. Reoviridae Reovirus, RotavirusNaked Icosahedral ds III 2. Picornaviridae Enterovirus, Rhinovirus,Naked Icosahedral ss IV Hepatovirus, Cardiovirus, Aphthovirus,Poliovirus, Parechovirus, Erbovirus, Kobuvirus, Teschovirus, Coxsackie3. Caliciviridae Norwalk virus Naked Icosahedral ss IV 4. TogaviridaeRubella virus Enveloped Icosahedral ss IV 5. Arenaviridae LymphocyticEnveloped Complex ss(—) V choriomeningitis virus 6. Flaviviridae Denguevirus, Hepatitis C Enveloped Icosahedral ss IV virus, Yellow fever virus7. Orthomyxoviridae Influenzavirus A, Enveloped Helical ss(—) VInfluenzavirus B, Influenzavirus C, Isavirus, Thogotovirus 8.Paramyxoviridae Measles virus, Mumps virus, Enveloped Helical ss(—) VRespiratory syncytial virus, Rinderpest virus, Canine distemper virus 9.Bunyaviridae California encephalitis virus, Enveloped Helical ss(—) VHantavirus 10. Rhabdoviridae Rabies virus Enveloped Helical ss(—) V 11.Filoviridae Ebola virus, Marburg virus Enveloped Helical ss(—) V 12.Coronaviridae Corona virus Enveloped Helical ss IV 13. AstroviridaeAstrovirus Naked Icosahedral ss IV 14. Bornaviridae Borna disease virusEnveloped Helical ss(—) V 15. Arteriviridae Arterivirus, EquineArteritis Enveloped Icosahedral ss IV Virus 16. Hepeviridae Hepatitis Evirus Naked Icosahedral ss IV

Retroviruses may also be affected:

-   -   Genus Alpharetrovirus; type species: Avian leukosis virus;        others include Rous sarcoma virus    -   Genus Betaretrovirus; type species: Mouse mammary tumour virus    -   Genus Gammaretrovirus; type species: Murine leukemia virus;        others include Feline leukemia virus    -   Genus Deltaretrovirus; type species: Bovine leukemia virus;        others include the cancer-causing Human T-lymphotropic virus    -   Genus Epsilonretrovirus; type species: Walleye dermal sarcoma        virus    -   Genus Lentivirus; type species: Human immunodeficiency virus 1;        others include Simian, Feline immunodeficiency viruses    -   Genus Spumavirus; type species: Simian foamy virus    -   Family Hepadnaviridae—e.g. Hepatitis B virus

Other viral diseases that may be affected by administration of theantibodies include Colorado Tick Fever (caused by Coltivirus, RNAvirus), West Nile Fever (encephalitis, caused by a flavivirus thatprimarily occurs in the Middle East and Africa), Yellow Fever, Rabies(caused by a number of different strains of neurotropic viruses of thefamily Rhabdoviridae), viral hepatitis, gastroenteritis (viral)-acuteviral gastroenteritis caused by Norwalk and Norwalk-like viruses,rotaviruses, caliciviruses, and astroviruses, poliomyelitis, influenza(flu), caused by orthomyxoviruses that can undergo frequent antigenicvariation, measles (rubeola), paramyxoviridae, mumps, Respiratorysyndromes including viral pneumonia and acute respiratory syndromesincluding croup caused by a variety of viruses collectively referred toas acute respiratory viruses, and respiratory illness caused by therespiratory syncytial virus (RSV, the most dangerous cause ofrespiratory infection in young children).

In some embodiments, the anti-DNA antibodies can be used in combinationwith radiotherapy, chemotherapy, or a combination thereof, to treat anycancer, including carcinomas, gliomas, sarcomas, or lymphomas. In theseembodiments, the anti-DNA antibodies can sensitize the cells to theDNA-damaging effects of radiotherapy or chemotherapy. A representativebut non-limiting list of cancers that the antibodies can be used totreat include cancers of the blood and lymphatic system (includingleukemias, Hodgkin's lymphomas, non-Hodgkin's lymphomas, solitaryplasmacytoma, multiple myeloma), cancers of the genitourinary system(including prostate cancer, bladder cancer, renal cancer, urethralcancer, penile cancer, testicular cancer), cancers of the nervous system(including mengiomas, gliomas, glioblastomas, ependymomas) cancers ofthe head and neck (including squamous cell carcinomas of the oralcavity, nasal cavity, nasopharyngeal cavity, oropharyngeal cavity,larynx, and paranasal sinuses), lung cancers (including small cell andnon-small cell lung cancer), gynecologic cancers (including cervicalcancer, endometrial cancer, vaginal cancer, vulvar cancer ovarian andfallopian tube cancer), gastrointestinal cancers (including gastric,small bowel, colorectal, liver, hepatobiliary, and pancreatic cancers),skin cancers (including melanoma, squamous cell carcinomas, and basalcell carcinomas), breast cancer (including ductal and lobular cancer),and pediatric cancers (including neuroblastoma, Ewing's sarcoma, Wilmstumor, medulloblastoma).

In some embodiments, the cancer is a neoplasm or tumor that demonstratessome resistance to radiotherapy or chemotherapy. Cancers that areresistant to radiotherapy using standard methods include, but are notlimited to, sarcomas, renal cell cancer, melanoma, lymphomas, leukemias,carcinomas, blastomas, and germ cell tumors.

C. Radiotherapy

The disclosed cell-penetrating anti-DNA antibodies can be used incombination with radiation therapy, Radiation therapy (a.k.a.radiotherapy) is the medical use of ionizing radiation as part of cancertreatment to control malignant cells. Radiotherapy also has severalapplications in non-malignant conditions, such as the treatment oftrigeminal neuralgia, severe thyroid eye disease, pterygium, pigmentedvillonodular synovitis, prevention of keloid scar growth, and preventionof heterotopic ossification. In some embodiments, anti-DNA antibodiesare used to increase radiosensitivity for a non-malignant condition.

Radiation therapy works by damaging the DNA of dividing cells, e.g.,cancer cells. This DNA damage is caused by one of two types of energy,photon or charged particle. This damage is either direct or indirect.Indirect ionization happens as a result of the ionization of water,forming free radicals, notably hydroxyl radicals, which then damage theDNA. For example, most of the radiation effect caused by photon therapyis through free radicals. One of the major limitations of photonradiotherapy is that the cells of solid tumors become deficient inoxygen, and tumor cells in a hypoxic environment may be as much as 2 to3 times more resistant to radiation damage than those in a normal oxygenenvironment.

Direct damage to cancer cell DNA occurs through high-LET (linear energytransfer) charged particles such as proton, boron, carbon or neon ions.This damage is independent of tumor oxygen supply because theseparticles act mostly via direct energy transfer usually causingdouble-stranded DNA breaks. Due to their relatively large mass, protonsand other charged particles have little lateral side scatter in thetissue; the beam does not broaden much, stays focused on the tumor shapeand delivers small dose side-effects to surrounding tissue. The amountof radiation used in photon radiation therapy is measured in Gray (Gy),and varies depending on the type and stage of cancer being treated. Forcurative cases, the typical dose for a solid epithelial tumor rangesfrom 60 to 80 Gy, while lymphomas are treated with 20 to 40 Gy.Post-operative (adjuvant) doses are typically around 45-60 Gy in 1.8-2Gy fractions (for breast, head, and neck cancers). Many other factorsare considered by radiation oncologists when selecting a dose, includingwhether the patient is receiving chemotherapy, patient co-morbidities,whether radiation therapy is being administered before or after surgery,and the degree of success of surgery.

The response of a cancer to radiation is described by itsradiosensitivity. Highly radiosensitive cancer cells are rapidly killedby modest doses of radiation. These include leukemias, most lymphomasand germ cell tumors. The majority of epithelial cancers are onlymoderately radiosensitive, and require a significantly higher dose ofradiation (60-70 Gy) to achieve a radical cure. Some types of cancer arenotably radioresistant, that is, much higher doses are required toproduce a radical cure than may be safe in clinical practice. Renal cellcancer and melanoma are generally considered to be radioresistant.

The response of a tumor to radiotherapy is also related to its size. Forcomplex reasons, very large tumors respond less well to radiation thansmaller tumors or microscopic disease. Various strategies are used toovercome this effect. The most common technique is surgical resectionprior to radiotherapy. This is most commonly seen in the treatment ofbreast cancer with wide local excision or mastectomy followed byadjuvant radiotherapy. Another method is to shrink the tumor withneoadjuvant chemotherapy prior to radical radiotherapy. A thirdtechnique is to enhance the radiosensitivity of the cancer by givingcertain drugs during a course of radiotherapy. The disclosedcell-penetrating anti-DNA antibodies serve this third function. In theseembodiments, the anti-DNA antibody increases the cell's sensitivity tothe radiotherapy, for example, by at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%. Moreover, the cell penetrating anti-DNA antibodies can becombined with one or more additional radiosensitizers. Examples of knownradiosensitizers include cisplatin, gemcitabine, 5-fluorouracil,pentoxifylline, vinorelbine, PARP inhibitors, histone deacetylaseinhibitors, and proteasome inhibitors.

D. Chemotherapeutics

Numerous chemotherapeutics, especially antineoplastic drugs, areavailable for combination with the antibodies. The majority ofchemotherapeutic drugs can be divided into alkylating agents,antimetabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, monoclonal antibodies, and other antitumour agents.

In preferred embodiments, the antineoplastic drug damages DNA orinterferes with DNA repair since these activities will synergize mosteffectively with the anti-DNA antibody. In these embodiments, theantibody increases the cell's sensitivity to the chemotherapy, forexample, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.Non-limiting examples of antineoplastic drugs that damage DNA or inhibitDNA repair include carboplatin, carmustine, chlorambucil, cisplatin,cyclophosphamide, dacarbazine, daunorubicin, doxorubicin, epirubicin,idarubicin, ifosfamide, lomustine, mechlorethamine, mitoxantrone,oxaliplatin, procarbazine, temozolomide, and valrubicin. In someembodiments, the antineoplastic drug is temozolomide, which is a DNAdamaging alkylating agent commonly used against glioblastomas. In someembodiments, the antineoplastic drug is a PARP inhibitor, which inhibitsa step in base excision repair of DNA damage. In some embodiments, theantineoplastic drug is a histone deacetylase inhibitor, which suppressesDNA repair at the transcriptional level and disrupt chromatin structure.In some embodiments, the antineoplastic drug is a proteasome inhibitor,which suppresses DNA repair by disruption of ubiquitin metabolism in thecell. Ubiquitin is a signaling molecule that regulates DNA repair. Insome embodiments, the antineoplastic drug is a kinase inhibitor, whichsuppresses DNA repair by altering DNA damage response signalingpathways.

In other embodiments, the antineoplastic drug complements the anti-DNAantibodies by targeting a different activity in the cancer cell. Inthese embodiments, the antineoplastic drug does not inhibit DNA repairor damage DNA.

Examples of antineoplastic drugs that can be combined with thecell-penetrating anti-DNA antibodies include, but are not limited to,alkylating agents (such as temozolomide, cisplatin, carboplatin,oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil,dacarbazine, lomustine, carmustine, procarbazine, chlorambucil andifosfamide), antimetabolites (such as fluorouracil, gemcitabine,methotrexate, cytosine arabinoside, fludarabine, and floxuridine), someantimitotics, and vinca alkaloids such as vincristine, vinblastine,vinorelbine, and vindesine), anthracyclines (including doxorubicin,daunorubicin, valrubicin, idarubicin, and epirubicin, as well asactinomycins such as actinomycin D), cytotoxic antibiotics (includingmitomycin, plicamycin, and bleomycin), and topoisomerase inhibitors(including camptothecins such as irinotecan and topotecan andderivatives of epipodophyllotoxins such as amsacrine, etoposide,etoposide phosphate, and teniposide).

E. Pharmaceutical Compositions

The cell-penetrating anti-DNA antibodies can be used therapeutically incombination with a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition is a unit dosage containinga cell-penetrating anti-DNA antibody or fragment thereof in apharmaceutically acceptable excipient, wherein the antibody is presentin an amount effective to inhibit DNA repair in a cancer or infectedcell. In preferred embodiments, the antibody is present in amount fromabout 200 mg/m² to about 1000 mg/m², more preferably about 200, 250,300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg/m². In someembodiments, the unit dosage is in a unit dosage form for intravenousinjection. In some embodiments, the unit dosage is in a unit dosage formfor intratumoral injection.

The materials may be in solution, emulsions, or suspension (for example,incorporated into microparticles, liposomes, or cells). Typically, anappropriate amount of a pharmaceutically-acceptable salt is used in theformulation to render the formulation isotonic. Examples ofpharmaceutically-acceptable carriers include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Pharmaceutical compositions may include carriers,thickeners, diluents, buffers, preservatives, and surface active agents.Further carriers include sustained release preparations such assemi-permeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped particles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered. Pharmaceuticalcompositions may also include one or more active ingredients such asantimicrobial agents, anti-inflammatory agents, and anesthetics.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases.

To aid dissolution of antibodies into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrosefatty acid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios. Additiveswhich potentially enhance uptake of peptides are for instance the fattyacids oleic acid, linoleic acid and linolenic acid.

III. Methods

A. Therapeutic Administration

Methods are provided for treating cancer or an infection in a subject byadministering to the subject a therapeutically effective amount ofcell-penetrating anti-DNA antibodies. Also provided are methods ofincreasing a cell's radiosensitivity or chemosensitivity in a subject byadministering to the subject a pharmaceutical composition containingcell-penetrating anti-DNA antibodies. In some embodiments, the methodinvolves first selecting a subject that has been diagnosed with aneoplasm, such as a cancer or tumor, or an infection with a pathogensuch as a virus.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. For example, the compositions may be administeredintravenously, intramuscularly, intrathecally, intraperitoneally,subcutaneously. The compositions may be administered directly into atumor or tissue, e.g., stereotactically. In some embodiments, thecompositions are administered into the brain or liver by injection or bya surgically implanted shunt.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the disorder being treated, and its mode ofadministration. Thus, it is not possible to specify an exact amount forevery composition. However, an appropriate amount can be determined byone of ordinary skill in the art using only routine experimentationgiven the teachings herein. For example, effective dosages and schedulesfor administering the compositions may be determined empirically, andmaking such determinations is within the skill in the art. The dosageranges for the administration of the compositions are those large enoughto impair DNA repair in target cells and/or sensitize the target cellsto radiotherapy and/or chemotherapy. The dosage should not be so largeas to cause adverse side effects, such as unwanted cross-reactions,anaphylactic reactions, and the like. Generally, the dosage will varywith the age, condition, and sex of the patient, route ofadministration, whether other drugs are included in the regimen, and thetype, stage, and location of the cancer to be treated. The dosage can beadjusted by the individual physician in the event of anycounter-indications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. A typical daily dosage of the antibody usedalone might range from about 1 μg/kg to up to 100 mg/kg of body weightor more per day, depending on the factors mentioned above. In preferredembodiments, the antibody is present in amount from about 200 mg/m² toabout 1000 mg/m², more preferably from about 200-900, 300-800, 400-700,500-600 mg/m². In some embodiments, the unit dosage is in a unit dosageform for intravenous injection. In some embodiments, the unit dosage isin a unit dosage form for oral administration. In some embodiments, theunit dosage is in a unit dosage form for intratumoral injection.

The anti-DNA antibodies increase a cancer's radiosensitivity orchemosensitivity. Effective doses of chemotherapy and/or radiationtherapy may be toxic for certain cancers. In some embodiments, theanti-DNA antibodies decrease the required effective dose of ananti-neoplastic drug or radiation levels needed to treat a cancer,thereby reducing toxicity of the effective dose. For example, the mostcommonly used dosage of doxorubicin is 40 to 60 mg/m² IV every 21 to 28days, or 60 to 75 mg/m² IV once every 21 days. If the patient has abilirubin level between 1.2 and 3 mg/dL, the dose should be reduced by50%. If the patient has a bilirubin level between 3.1 and 5.0 mg/dL, thedose should be reduced by 75%. Serious irreversible myocardial toxicityleading to congestive heart failure often unresponsive to cardiacsupport therapy may be encountered as the total dosage of doxorubicinapproaches 450 mg/m². When used in combination with the anti-DNAantibodies, doxorubicin dosage may be reduced to decrease myocardialtoxicity without a loss in efficacy.

In other embodiments, the disclosed anti-DNA antibodies may be used withnormal doses of drug or radiation to increase efficacy. For example, theanti-DNA antibodies may be used to potentiate a drug or radiationtherapy for a cancer that is drug or radiation resistant. Cancers thatare resistant to radiotherapy using standard methods include sarcomas,lymphomas, leukemias, carcinomas, blastomas, and germ cell tumors.

B. Screening Assay

Since anti-DNA antibodies are shown to inhibit DNA repair and increaseradiosensitivity and/or chemosensitivity in cancer cells, a method ofdetecting anti-DNA antibodies in a sample is also provided. For example,the sample can be a bodily fluid containing antibodies, such as blood,serum, or plasma from a subject having, or suspected of having, SLE. Thesample can be a tissue or cell sample, such as a biopsy.

In some embodiments, the method can be used to monitor the diagnosis orprognosis of a subject with SLE. For example, detection of cellpenetrating anti-DNA antibodies can provide early detection of patientsabout to undergo an SLE flare-up.

In some embodiments, the method can be used to predict a subject'ssensitivity to chemotherapy or radiotherapy. In these embodiments, thelevels of cell penetrating anti-DNA antibodies in the sample cancorrespond to the level of sensitivity to chemotherapy or radiotherapy.In preferred embodiments, the chemotherapy is one that inhibits DNArepair or causes DNA damage.

The method can involve contacting cells with the sample from the subjectand monitoring the effect of the sample on the cells. The cells arepreferably a cell line, such as a cancer cell line, that has been shownto normally be radioresistant or chemoresistant but after treatment withanti-DNA antibodies, become radiosensitive and/or chemosensitive. Insome embodiments, the cells are irradiated, and the method involvesevaluating the effect of the sample on cell radiosensitivity. In otherembodiments, the cells are contacted with an anti-neoplastic drug, andthe method involves evaluating the effect of the sample onchemosensitivity. In still other embodiments, the method involvesmonitoring the direct effect of the sample on cell death. In all ofthese embodiments, an increase in radiosensitivity, chemosensitivity, orcell death, is an indication that the sample contains anti-DNAantibodies.

In other embodiments, the method involves an immunoassay, such as anELISA or flow cytometry designed to detect anti-DNA antibodies. Inpreferred embodiments, the method is cell-based to detect cellpenetration.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1: A Cell-Penetrating Anti-DNA Antibody (3E10) EnhancesCellular Radiosensitivity In Vitro

Materials and Methods

Cell Lines:

MCF-7, HeLa, U251, and U87 cell lines were obtained from the AmericanType Culture Collection (ATCC). PEO1 and PEO1 C4-2 cells were obtained(Sakai W, et al. Cancer Res 69:6381 (2009)). Cells were grown andmaintained in Dulbecco's Modification of Eagles Medium (DMEM;Mediatech®) supplemented with 10% fetal bovine serum (FBS) at 37° C. in5% CO₂.

Production and Purification of 3E10 and 3E10 scFv:

3E10 was purified from hybridoma supernatant as described by Weisbart RH, et al. J Immunol 144; 2653 (1990). 3E10 scFv was expressed in Pichiapastoris and was purified from supernatant as described by Hansen J E,et al. Brain Res 1088:187 (2006). Protein concentrations were determinedby Bradford assay.

In Vitro Cell Survival Assays:

Clonogenic assays and propidium iodide based assays were performed asdescribed Hansen J E, et al. Cancer Res 67:1769 (2007); Stachelek G C,et al. Cancer Res 70:409 (2010)).

Cellular Irradiation:

Cells grown in 6- or 12-well plates were irradiated with x-rays at thedoses specified using the X-RAD 320 Biological Irradiator (PrecisionX-Ray) in accordance with the manufacturer's instructions.

Table 1 presents data demonstrating that 3E10 scFv sensitizes MCF-7 andHeLa cells to IR. MCF-7 and Hela cells were irradiated in the presenceof media containing control buffer or 3E10 scFv, and clonogenic survivalwas determined by colony formation assay. Surviving fraction±standarderror of the mean relative to unirradiated control cells are presentedfor each treatment. Dose of 3E10 scFv was 0.25 μM for MCF-7 cells and1.0 μM for HeLa cells. IR dose was 4 Gy for MCF-7 cells and 6 Gy forHeLa cells.

Results

SLE is an autoimmune disease characterized by aberrant production ofautoantibodies reactive against host DNA (anti-nuclear antibodies;ANAs). A rare subset of these antibodies can penetrate into cells, butalmost all are cytotoxic and inappropriate for clinical use(Alarcon-Segovia, D. Lupus 10:315 (2001)). However, one unusualcell-penetrating anti-DNA antibody, 3E10, isolated from a mouse model ofSLE, was not found to be toxic to cells in culture or to mice in vivo,and was even shown to be safe in a Phase I human clinical trialevaluating the potential use of 3E10 in a vaccine to treat SLE (SpertiniF, et al. J Rheumatol 26:2602 (1999); Weisbart R H, et al. J Immunol144:2653 (1990); Zack D J, et al. J Immunol 157:2082 (1996)). 3E10 wasnot further pursued as a vaccine but was instead investigated as amolecular delivery vehicle (Hansen J E, et al. Brain Res 1088:187(2006); Hansen J E, et al. J Biol Chem 282:20790 (2007)).

Besides its benign toxicity profile, 3E10 is further distinguished fromother cell-penetrating ANAs by its mechanism of cellular penetration.Unlike all the others, cell penetration by 3E10 is independent of its Fcor constant domains; rather, the 3E10 single chain variable fragment(3E10 scFv) can, by itself, penetrate cells and localize in nuclei), ina mechanism mediated by an equilibrative nucleoside transporter that isubiquitous on human cells (Hansen J E, et al. J Biol Chem 282:20790(2007); Lisi S et al. Clin Exp Med 11:1 (2011)).

Both the full 3E10 antibody and 3E10 scFv have proven capable ofdelivering cargo proteins such as p53 and Hsp70 into cells in vitro andin vivo (Hansen J E, et al. Brain Res 1088:187 (2006); Hansen J E, etal. Cancer Res 67:1769 (2007); Zhan X, et al. Stroke 41:538 (2010)).“3E10 scFv” utilized in the experiments disclosed herein is the moleculedescribed in Hansen J E, et al. J Biol Chem 282:20790 (2007), andcitations referenced therein, which includes an antibody 3E10 VL domainlinked to a 3E10 VH domain with a D31N mutation reported to enhance DNAbinding and cell penetration. It was intended to use 3E10 to transportlinked molecules with known radiosensitizing effects into cancer cellsto enhance tumor response to radiotherapy. However, in initialexperiments, it was discovered that 3E10, by itself, enhances cellularradiosensitivity. This is shown in Table 1 and FIG. 1B. Table 1 presentsdata demonstrating that 3E10 scFv sensitizes MCF-7 and HeLa cells to IR.MCF-7 and Hela cells were irradiated in the presence of media containingcontrol buffer or 3E10 scFv, and clonigenic survival was determined bycolony formation assay. Surviving fraction±standard error of the meanrelative to unirradiated control cells are presented for each treatment.Dose of 3E10 scFv was 0.25 μM for MCF-7 cells and 1.0 μM for HeLa cells.IR dose was 4 Gy for MCF-7 cells and 6 Gy for HeLa cells.

FIG. 1B shows a clonogenic survival assay measuring the impact of 3E10scFv on the survival of U251 human glioma cells treated with ionizingradiation (IR). U251 cells were incubated with growth media containing3E10 scFv or control buffer for one hour, and cells were then irradiatedwith 0 or 4 Gyof IR and evaluated for clonogenicity by colony formation.Consistent with prior studies, the 3E10 scFv by itself was not toxic tounirradiated U251 cells. However, U251 cells irradiated in the presenceof 3E10 scFv were found to be more sensitive to IR. 3E10 scFv alsoincreased the radiosensitivity of MCF-7 human breast cancer cells andHeLa human cervical cancer cells at doses as low as 0.25 μM (Table 1).Radiosensitization by a cell-penetrating, anti-DNA antibody has not beenpreviously described.

Example 2: A Cell-Penetrating Anti-DNA Antibody (3E10) PotentiatesDNA-Damaging Chemotherapy In Vitro

Materials and Methods

Since radiation targets DNA, the impact of 3E10 on cancer cell responseto DNA-damaging chemotherapy was tested. Specifically, the influence of3E10 on cell sensitivity to doxorubicin versus paclitaxel, two agentscommonly used in cancer therapy, was compared. Doxorubicin is ananthracycline antibiotic that intercalates into DNA and induces strandbreaks (Tewey K M, et al. Science 226:466 (1984)), while paclitaxelinterferes with microtubule function (Jordan M A, et al. Proc Natl AcadSci USA 90:9552 (1993)) but does not directly damage DNA.

It was predicted that 3E10 scFv would sensitize cells to doxorubicin butnot to paclitaxel. U87 human glioma cells were treated with increasingdoses of doxorubicin (0-250 nM) or paclitaxel (0-25 nM) in the presenceof control buffer or 10 μM 3E10 scFv, and percent cell killing was thendetermined by propidium iodide fluorescence one week after treatment.

Results

As predicted, 3E10 scFv significantly enhanced the sensitivity of thecells to doxorubicin but not to paclitaxel (FIGS. 1C and 1D). Theability of 3E10 scFv to sensitize cancer cells to both IR anddoxorubicin but not to paclitaxel indicates that the antibodyselectively potentiates cell killing by DNA-damaging therapies.

Example 3: A Cell-Penetrating Anti-DNA Antibody (3E10) Inhibits DNARepair

Upon establishing that 3E10 scFv sensitizes cells to DNA-damagingagents, the mechanism underlying this effect was investigated. Both IRand doxorubicin induce DNA strand breaks, and so it was hypothesizedthat 3E10 scFv might have an effect on DNA repair, particularly strandbreak repair. As a first step, the DNA binding properties of 3E10 wereexamined. The binding affinity of 3E10 for several different DNAsubstrates, including single-stranded DNA, blunt-end duplex DNA, duplexDNA with an internal bubble due to heterology, duplex DNA with splayedsingle-stranded ends, duplex DNA with a 5′ single-stranded tail, andduplex DNA with a 3′ tail, (FIG. 2A) was determined by incubatingradiolabeled DNA substrates prepared as described by Xu X, et al. EMBO J28:568 (2009) with increasing concentrations of 3E10 (0-1 μM) followedby electrophoretic mobility shift analyses.

Materials and Methods

DNA Binding Studies:

Radiolabeled DNA substrates (single-stranded DNA, blunt-end duplex DNA,duplex DNA with an internal bubble due to heterology, duplex DNA withsplayed single-stranded ends, duplex DNA with a 5′ single-stranded tail,and duplex DNA with a 3′ tail) were prepared as described by Xu X, etal. EMBO J 28:3005 (2009). Each substrate was incubated with 3E10 (0-10μM) for 30 minutes at 4° C., followed by electrophoretic mobility shiftanalysis as described by Xu X, et al. EMBO J 28:3005 (2009). Kd wascalculated by plotting percent oligonucleotide bound using ImageJ;National Institutes of Health versus concentration of 3E10.

DNA Repair Assays:

Single-strand break/base excision repair (BER) and RAD51-mediated strandexchange assays were performed as described by, respectively Stachelek GC, et al. Cancer Res 70:409 (2010); and Dray E, et al. Proc Natl AcadSci USA 108:3560 (2011).

Microscopy:

Immunohistochemistry and γH2AX immunofluorescence were performed aspreviously described (Hansen J E, et al. J Biol Chem 282:20790 (2007);Stachelek G C, et al. Cancer Res 70:409 (2010)).

Results

A left-shift in the binding curves for the single-strand, splayed arm,and 5′/3′ tail substrates relative to those for the duplex and bubblesubstrates was observed, suggesting that 3E10 binds with greateraffinity to substrates with free single-strand tails (FIGS. 2C-2H). Theresults were combined and plotted to directly compare the binding of3E10 to substrates with or without a free single-strand tail. Overall,3E10 bound to substrates with a free single-strand tail with a K_(d) of0.2 μM and to substrates without a free single-strand tail with a K_(d)of 0.4 μM (FIG. 2B). These results suggest that upon cellularpenetration and nuclear localization, 3E10 may preferentially bind toDNA repair or replication intermediates that consist of duplex DNA withsingle-stranded tails.

The impact of 3E10 on specific DNA repair pathways was next examined,starting with an in vitro single-strand break/base excision repair (BER)assay (Stachelek G C, et al. Cancer Res 70:409 (2010)). In BER, adamaged base is excised by a glycosylase followed by cleavage of thephosphodiester backbone by an endonuclease to yield a substrate with asingle-strand break (product n). The dRP lyase activity of DNApolymerase removes the dRP group, and its polymerase activity insertsthe missing nucleotide and restores correct base pairing (product n+1).This is followed by ligation of the residual strand break by ligase torestore the integrity of the phosphodiester backbone, leading toconversion of the n+1 product into the full-length product in duplexconformation. The efficiency of BER may therefore be determined bytracking the levels of the n and n+0.1 species over time as quantifiedrelative to the percentage of total substrate and product DNA. Tomeasure the impact of 3E10 on BER, the repair of a mismatch in aradiolabeled DNA substrate (incubated with uracil DNA glycosylase, APendonuclease, DNA polymerase β, and ligase) was tested in the presenceof control buffer, control anti-tubulin antibody, or 3E10. 3E10significantly delayed the conversion of the n+1 species to the finalligated product relative to the buffer control, suggesting that 3E10impairs the ligation step of the single-strand break repair pathway(FIG. 2I). The anti-tubulin antibody had no effect on BER relative tocontrol buffer.

To further probe the impact of 3E10 on DNA repair the effect of 3E10 onhomology-dependent repair (HDR), a key pathway for the repair of DNAdouble-strand breaks (DSB), including IR-induced DSBs and DSBsassociated with stalled replication forks, was tested (Arnaudeau C, etal. J Mol Biol 307:1235 (2001); Li X, et al. Cell Res 18:99 (2008)). HDRis dependent on the RAD51 recombinase, which binds single-strand DNA toform nucleofilaments that mediate strand invasion (Sung P, et al.Science 265:1241 (1994); Sung P, et al. J Biol Chem 278:42729 (2003);Sung P, et al. Cell 82:453 (1995)). Since 3E10 preferentially binds freesingle-strand tails it was hypothesized that the antibody might impairRAD51-mediated strand invasion and exchange. This hypothesis was testedin an in vitro strand exchange assay (Dray E, et al. Proc Natl Acad SciUSA 108:3560 (2011)), the schematic for which is shown in FIG. 3A.Purified human RAD51 (hRAD51) was incubated for 5 minutes with anunlabeled 150 bp single-stranded DNA substrate to allow formation of anhRAD51-single-stranded DNA nucleoprotein capable of strand invasion.3E10 (0-35 μM) or a control anti-His₆ IgG antibody was next added to thereaction and allowed to incubate for 5 minutes. Next, a 40 bp DNAsubstrate in blunt end duplex conformation with one radiolabeled strandwas added and allowed to incubate with the hRAD51-single-stranded DNAnucleoprotein filament in the presence or absence of antibody for 30minutes (schematic shown in FIG. 3A). The degree of strand exchange wasthen visualized by electrophoresis and quantified. 3E10 inhibited strandexchange in a dose-dependent manner (FIG. 3B), while the controlanti-His₆ IgG antibody had no impact on strand exchange. 3E10 was alsoable to suppress strand exchange mediated by a RAD51 variant,hRAD51K133R, that is defective in ATP hydrolysis and so is an even morepotent mediator of strand exchange than wild-type RAD51 (Chi P, et al.DNA Repair (Amst) 5:381 (2006)) (FIG. 3C). These data demonstrateinhibition of HDR by 3E10 via suppression of RAD51-mediated strandexchange.

Since 3E10 was found to inhibit HDR in vitro, it was hypothesized thatrepair of DNA DSBs induced by DNA-damaging therapy would be delayed incells treated with 3E10. To test this hypothesis, U251 glioma cells weretreated with control buffer or 3E10 scFv (10 μM) and irradiated with 2Gy of IR. Twenty-four hours later, the numbers of persisting DNA DSBsper cell were quantified by visualization of foci of the phosphorylatedhistone component, γH2AX, via immunofluorescence. Cells irradiated inthe presence of 3E10 scFv showed an average of 10.5±1 γH2AX foci percell at 24 hours, compared to 6.8±1 in cells irradiated in controlbuffer (p<0.01). These data demonstrate delayed resolution of DNA DSBsin cells treated with 3E10 scFv, in keeping with the in vitro resultsdemonstrating inhibition of DNA repair by 3E10.

Example 4: A Cell-Penetrating Anti-DNA Antibody (3E10) is SyntheticallyLethal to Cancer Cells Deficient in DNA Repair

Materials and Methods

Cancer cells harboring deficiencies in HDR due to BRCA2 mutations(Moynahan M E, et al. Mol Cell 7:263 (2001)) are highly vulnerable tokilling by inhibition of single-strand break repair (Bryant H E, et al.Nature 434:913 (2005); Feng Z, et al. Proc Natl Acad Sci USA 108:686(2011); Kaelin, Jr. W G, et al. Nat Rev Cancer 5:689 (2005)). Suchinhibition can be achieved by treatment with inhibitors ofpoly(ADP-ribose) polymerase 1 (PARP-1) or DNA polymerase β, a phenomenontermed synthetic lethality (Stachelek G C, et al, Cancer Res 70:409(2010); Bryant H E, et al. Nature 434:913 (2005); Kaelin, Jr. W G, etal. Nat Rev Cancer 5:689 (2005); Farmer H, et al. Nature 434:917(2005)). In addition, BRCA2-deficient cells have been shown to besensitive to further inhibition of HDR as shown by the synthetic lethaleffect of knockdown of RAD52 in BRCA2-deficient cancer cells (Feng Z, etal. Proc Natl Acad Sci USA 108:686 (2011)). Based on the observationthat 3E10 inhibits both single-strand break repair and HDR, it washypothesized that 3E10 would be synthetically lethal to BRCA2-deficientcancer cells. To test this hypothesis, the impact of treatment with 3E10scFv or the full 3E10 antibody on a matched pair of BRCA2-deficient(PEO1) and BRCA2-proficient (PEO1 C4-2) human ovarian cancer cells wasevaluated (Sakai W, et al. Cancer Res 69:6381 (2009)). The impact of3E10 scFv was also tested on BRCA2-proficient PEO4 human ovarian cancercells and BRCA2-deficient CAPAN1/neo human pancreatic cancer cells.

Results

3E10 scFv TREATMENT reduced the clonogenic survival of theBRCA2-deficient PEO1 cells (p=0.03) but did not adversely impact thesurvival of the BRCA2-proficient PEO1 C4-2 cells (FIG. 4A-4B). 3E10 scFvwas also toxic to BRCA2-deficient PEO1 cells but BRCA2-proficient PEO4cells in a cell-viability assay (FIG. 4C). 3E10 scFv alone was alsotoxic to BRCA2-deficient human pancreatic cancer cells (CAPAN1/neo)(FIG. 4D). The full 3E10 antibody was similarly selectively toxic to theBRCA2-deficient PEO1 cells in a dose-dependent manner (FIG. 4E). Thesedata provide the first evidence for a synthetic lethal effect of acell-penetrating anti-DNA antibody on cancer cells deficient in DNArepair and demonstrate the potential utility of 3E10 as a targetedcancer therapy for malignancies with DNA repair deficiencies.Importantly, the synthetic lethal effect of 3E10 on BRCA2-deficientcancer cells is in keeping with the mechanistic experiments describedabove showing that 3E 10 impacts DNA strand break repair.

In addition, hypoxic cancer cells have been shown to have reducedhomology=directed repair capacity (Bindra, et al. Mol. Cell. Biol.24(19):8504-8518 (2004)), so 3E10 and similar antibodies are expected tobe synthetically lethal to cancer cells that are hypoxic.

Example 5: A Cell-Penetrating Anti-DNA Antibody, 3E10, Profoundly andSelectively Sensitizes DNA-Repair Deficient Cancer Cells to DNA-DamagingTherapy

Materials and Methods

In order to determine whether 3E10 would have an even greater impact onBRCA2-deficient cancer cells when coupled with a DNA-damaging agent,BRCA2-deficient PEO1 and BRCA2-proficient PEO1 C4-2 ovarian cancer cellswere treated with control buffer, 10 μM 3E10, 3 nM doxorubicin, or 10 μM3E10+3 nM doxorubicin. Percent cell death was then evaluated bypropidium iodide fluorescence three days after treatment. A low dose ofdoxorubicin (3 nM) was selected to minimize the effect of doxorubicinalone on the cells, and the decision to measure percent cell death 3days after treatment was made in order to minimize the confoundingeffects of synthetic lethality by 3E10 alone on BRCA2-deficient cells,which is evident approximately seven days post-treatment.

Results

As expected, the low dose of doxorubicin alone was not significantlytoxic to BRCA2-proficient or BRCA2-deficient cells. The addition of 3E10to the doxorubicin had a minimal impact on the BRCA2-proficient cells.However, the combination of 3E10 and doxorubicin was highly cytotoxic toBRCA2-deficient cells (65%±7 cell death, p<0.001) (FIG. 4F-4G).

Example 6: 3E10 Potentiates DNA-Damaging Chemotherapy In Vivo

It was next determined whether the impact of 3E10 on tumor cellsensitivity to DNA-damaging therapy is preserved in vivo in a mousetumor model.

Materials and Methods

The full 3E10 antibody was used for in vivo studies due to its expectedgreater half-life in the circulation compared to the variable fragment.U87 glioma tumors were generated in SCID mice by subcutaneous injection.When tumors had formed and were in a consistent growth phase, mice weretreated by intraperitoneal injection of control buffer, 3E10 antibodyalone (0.8 mg in 0.5 mL PBS, 10 μM), doxorubicin alone (80 μg/kg), orboth 3E10 and doxorubicin. Each treatment group was composed of 4 mice.The impact of treatment was then evaluated by measuring tumor growththree days after injection. The selection of this time point for tumormeasurement was based on the in vitro studies that demonstrated that theantibody's impact on cancer cell sensitivity to doxorubicin can bedetected 3 days after treatment. Additional tumor measurements after 3days could not be obtained due to predetermined institutionalrequirements for animal sacrifice when tumors reached a volume of 400mm³, which was rapidly achieved in the control groups.

Results

Tumors in mice treated with control buffer increased in volume by54%±23. Treatment with 3E10 or doxorubicin alone did not significantlyimpact tumor growth, with tumor volumes increased by 48%±13 and 61%±10,respectively. By contrast, tumor growth was significantly reduced inmice treated with combined 3E10 and doxorubicin, with tumors increasedin size by only 9%±7 (p=0.004, with p calculated by comparison ofabsolute tumor volumes in mice treated with doxorubicin+3E10 versusdoxorubicin alone). These data demonstrate sensitization of tumors todoxorubicin by 3E10 in vivo (FIG. 5).

Example 7: A Cell-Penetrating Anti-DNA Antibody (3E10) Sensitizes HumanGlioma Xenografts to Ionizing Radiation In Vivo

Materials and Methods

Human glioma xenografts were generated by subcutaneous injection of U87cells into the flanks of nude mice. Treatment groups were: Control(n=8); Antibody (n=8), 8 Gy (of ionizing radiation) (n=8), and 8Gy+Antibody (n=7; one animal lost in anesthesia). Twenty-five days afterimplantation, tumors had grown to a mean size of ˜100 mm³ mice. On day26 the mice were treated with intraperitoneal injection of controlbuffer (PBS; “Control” and “8 Gy” groups) or 3E10 (1 mg in PBS;“Antibody” and “8 Gy+Antibody” groups). Each group then received asecond injection of the same reagent 24 hours later. 2 hours after thesecond injection tumors in the “8 Gy” and “8 Gy+Antibody” groups wereirradiated with 8 Gy. Tumor volumes in each group were then followed andmice were sacrificed when tumor volume reached 1000 mm³. Tumor growthmeasurements versus days after tumor implantation are shown in FIG. 6A.

Results

3E10 alone (open diamonds) had no significant impact on tumor growthrelative to control buffer alone (solid diamonds). However, thecombination of 3E 10 and 8 Gy (open triangles) suppressed tumor growthto a greater degree than 8 Gy alone (solid triangles).

In FIG. 6B, Kaplan-Meier plots of progression-free survival in eachgroup are presented. Progression-free survival is defined as survivalwith tumor not having increased in size by threefold or greater relativeto baseline size. Baseline size is defined as tumor size one day priorto antibody treatment, which is represented as day 25 in panel A and day0 in panel B. Tumor tripling time (time required for tumors to increasein volume threefold over baseline) was 9.5±0.5 days in tumors treatedwith 8 Gy as compared to 13.7±1.8 days in tumors treated with 8 Gy+3E10(p=0.04). 3E10 alone, however, had no impact on U87 tumors relative tocontrol buffer alone, with tumor tripling time of control tumors 6.8±0.7days versus 6.5±0.3 days in tumors treated with 3E10 alone (p=0.67).These data demonstrate sensitization of human glioma xenografts toionizing radiation by 3E10 in vivo. Error bars represent standard errorof the mean.

We claim:
 1. A method of inhibiting DNA repair in neoplastic or virallytransforming or virally transformed cells in a subject, comprisingadministering to the neoplastic, virally transforming or virallytransformed cells in the subject a pharmaceutical composition comprisingan effective amount of anti-DNA unconjugated antibodies or antigenbinding fragments thereof to impair a DNA repair pathway, wherein all ofthe anti-DNA unconjugated antibodies or antigen binding fragmentsthereof of the pharmaceutical composition consist of one or more cellpenetrating unconjugated monospecific anti-DNA antibodies or cellpenetrating unconjugated antigen binding fragments thereof, wherein thecell penetrating unconjugated monospecific anti-DNA antibodies or cellpenetrating unconjugated antigen binding fragments thereof consist ofmonoclonal antibody 3E10 produced by ATCC Accession No. PTA 2439hybridoma, or a cell penetrating antigen binding fragment or humanizedform thereof.
 2. The method of claim 1, wherein the neoplastic cells arecancer cells selected from the group consisting of sarcomas, lymphomas,leukemias, carcinomas and adenocarcinomas, blastomas, germ cell tumors,gliomas, neuroendocrine tumors, melanomas, rhabdoid tumors, embryonaltumors, neuroectodermal tumors, carcinoid tumors, craniopharyngiomas,histiocytomas, medulloepitheliomas, mesotheliomas, multiple myelomas,chronic myeloproliferative disease, primitive neuroectodermal tumors,salivary gland tumors, thymomas, thymic carcinoma, thyroid cancer, andWilms tumor.
 3. The method of claim 1, wherein the cells are radiationresistant.
 4. The method of claim 1, wherein the cells are resistant tochemotherapy.
 5. The method of claim 1, wherein the cells have intrinsicdefective or deficient DNA repair.
 6. The method of claim 1, wherein thevirally transforming or virally transformed cells are exposed to orinfected with a virus having or causing DNA repair defects ordeficiencies, or is dependent on host DNA repair pathways for infection,integration, or replication.
 7. The method of claim 6, wherein thevirally transforming or virally transformed cells are exposed to orinfected with a lentivirus.
 8. The method of claim 1, wherein the cellshave one or more mutations in or abnormal expression of DNA repair genesselected from the group consisting of XRCC1, ADPRT (PARP-1), ADPRTL2,(PARP-2), POLYMERASE BETA, CTPS, MLH1, MSH2, FANCD2, PMS2, p53, p21,PTEN, RPA, RPA1, RPA2, RPA3, XPD, ERCC1, XPF, WS19, RAD51, RAD51b,RAD51C, RAD51D, DMC1, XRCCR, XRCC3, BRCA1, BRCA2, PALB2, RAD52, RAD54,RAD50, MRE11, NB51, WRN, BLM, KU70, KU80, ATM, ATR CHK1, CHK2, FANCA,FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCC, FANCD1,FANCD2, FANCE, FANCF, FANCG, RAD1, and RAD9.
 9. The method of claim 1,wherein the cells have a defective tumor suppressor gene such as BRCA1or BRCA2.
 10. The method of claim 1, wherein the cells are part of ahypoxic tumor.
 11. The method of claim 1, wherein the antibodies areadministered to the subject in combination with a radiosensitizer. 12.The method of claim 11, wherein the radiosensitizer is selected from thegroup consisting of cisplatin, doxorubicin, gemcitabine, 5-fluorouracil,PARP1 inhibitors, histone deacetylase inhibitors, proteasome inhibitors,epidermal growth factor receptor (EGRF) inhibitors, insulin-like growthfactor-1 (IGF-1) receptor inhibitors, CHK1 inhibitors, mTOR inhibitors,kinase inhibitors, pentoxifylline, and vinorelbine.
 13. The method ofclaim 1, further comprising treating the subject with radiation therapy,wherein the cell-penetrating anti-DNA antibodies increase the cells'sensitivity to radiation therapy.
 14. The method of claim 1, furthercomprising treating the subject with an antineoplastic drug that damagesDNA or inhibits DNA repair, wherein the cell-penetrating anti-DNAantibodies increase the cells' sensitivity to the antineoplastic drug.15. The method of claim 1, further comprising treating the subject witha therapeutic monoclonal antibody.
 16. The method of claim 1, whereinthe cell-penetrating anti-DNA antibodies are derived from a subject withan autoimmune disease.
 17. The method of claim 16, wherein theautoimmune disease is systemic lupus erythematous, or an animal modelthereof.
 18. The method of claim 1, wherein the cell-penetratinganti-DNA antibodies or antigen binding fragments thereof consist of asingle chain variable fragment of the monoclonal antibody 3E10 producedby ATCC Accession No. PTA 2439 hybridoma or a humanized form thereof.19. The method of claim 1, wherein the cell-penetrating anti-DNAantibodies or antigen binding fragments thereof are transported into thenucleus of the cell without the aid of a carrier or conjugate.
 20. Themethod of claim 1, wherein administering the antibodies or antigenbinding fragments thereof in combination with chemotherapy or radiationtherapy increases DNA damage in the neoplastic or virally transformingor virally transformed cells caused by the radiation or chemotherapyrelative to administering of the radiation or chemotherapy alone. 21.The method of claim 1, wherein the antibodies or antigen bindingfragments thereof are not directly cytotoxic to DNA repair-proficientcells.
 22. The method of claim 21, wherein the antibodies or antigenbinding fragments are synthetically lethal to DNA repair-deficientcells.
 23. A method of treating a subject with cancer, comprisingadministering to the subject a pharmaceutical composition comprising aneffective amount of anti-DNA antibodies or antigen binding fragmentsthereof to impair a DNA repair pathway in cancer cells in the subject,wherein all of the anti-DNA unconjugated antibodies or antigen bindingfragments thereof of the pharmaceutical composition consist of one ormore cell penetrating unconjugated monospecific anti-DNA antibodies orcell penetrating unconjugated antigen binding fragments thereof, whereinthe cell penetrating unconjugated monospecific anti-DNA antibodies orantigen binding fragments thereof consist of monoclonal antibody 3E10produced by ATCC Accession No. PTA 2439 hybridoma, or a cell penetratingunconjugated antigen binding fragments or humanized form thereof. 24.The method of claim 23, further comprising administering to the subjecta DNA damaging agent.
 25. The method of claim 24, wherein the DNAdamaging agent is a chemotherapeutic drug or radiation.
 26. The methodof claim 23, wherein the subject comprises cancer cells that are DNArepair-deficient.
 27. The method of claim 26, wherein the DNArepair-deficient cells are BRCA1 or BRCA2 deficient.
 28. The method ofclaim 23, wherein the cell-penetrating anti-DNA antibodies or antigenbinding fragments thereof consist of a single chain variable fragment ofthe monoclonal antibody 3E10 produced by ATCC Accession No. PTA 2439hybridoma or a humanized form thereof.
 29. The method of claim 1,wherein the pharmaceutical composition comprises an effective amount ofthe unconjugated cell-penetrating anti-DNA antibodies or antigen bindingfragments thereof to reduce DNA repair.
 30. The method of claim 23,wherein the pharmaceutical composition comprises an effective amount ofthe unconjugated cell-penetrating anti-DNA antibodies or antigen bindingfragments thereof to reduce DNA repair.
 31. The method of claim 1,wherein the cell-penetrating anti-DNA antibodies or antigen bindingfragments thereof comprise a divalent single-chain variable fragment(discFv) of the monoclonal antibody 3E10 produced by ATCC Accession No.PTA 2439 hybridoma, or humanized form thereof.
 32. The method of claim23, wherein the cell-penetrating anti-DNA antibodies or antigen bindingfragments thereof comprise a divalent single-chain variable fragment(discFv) of the monoclonal antibody 3E10 produced by ATCC Accession No.PTA 2439 hybridoma, or humanized form thereof.